1. Field of the Invention
The present invention is concerned with methods of cloning and expressing the leukotoxin gene from Fusobacterium necrophorum (F. necrophorum), sequencing and characterizing the leukotoxin protein expressed by this gene, truncating the gene into a series of nucleotide sequences, amplifying these sequences, expressing and recovering the polypeptides encoded by the nucleotide sequences, and utilizing the protein and the polypeptides in recombinant vaccines in order to confer effective immunity against infection caused by the production of leukotoxin by F. necrophorum. More particularly, it is concerned with production of an inactivated recombinant leukotoxin vaccine generated by amplifying five leukotoxin gene fragments and one upstream region through PCR, digesting the nucleotide sequences encoded by the gene fragments with restriction enzymes, expressing the polypeptide sequences coded by the nucleotide sequences through an expression vector, recovering these proteins as five truncated leukotoxin proteins (or polypeptides), purifying these proteins (or polypeptides) to apparent homogeneity, with or without inactivation of the truncated and full length proteins, and combining the inactivated recombinant leukotoxins with adjuvants.
2. Description of the Prior Art
Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States. This estimate is based primarily on condemnation of liver and other organs, and does not include economic losses stemming from reduced feed intake, reduced feed efficiencies, decreased carcass dressing percentage and lowered weight gains. A number of studies have confirmed that cattle with abscessed livers gain less (average 4-5%) and have reduced feed efficiencies (average 7%) compared with cattle having healthy livers. The average incidence of abscessed liver in grain-fed cattle approximates 25-30%. To a lesser extent, liver abscesses in sheep and goats are also an economic problem.
F. necrophorum is a gram-negative, rod-shaped, nonsporeforming, nonmotile, strictly anaerobic and pleomorphic organism. Morphologically, the organism varies from short rods to filamentous with pointed and rounded ends. Cell lengths range from coccoid bodies of 0.5-0.7 xcexcm diameter to filaments over 100 xcexcm. Surface colonies are 1-2 mm in diameter, circular, transparent to opaque, and with some strains producing xcex1 or xcex2 hemolysis. The organism ferments glucose, fructose and maltose only weakly with final pH around 5.0-6.3. It ferments lactate to acetate, propionate, and butyrate. Butyrate is the major product from lactate fermentation. Indole is produced from peptone. F. necrophorum has been isolated from the normal flora in the oral cavity, gastrointestinal cavity, and genitourinary tract of humans and animals. The organism is also known to survive in the soil.
F. necrophorum is a normal inhabitant of the gastrointestinal tracts of animals and humans. Virulence factors and pathogenic mechanisms that contribute to the transition of this otherwise commensal organism to a pathogen are poorly understood. A leukotoxin, endotoxin, hemolysin, hemagglutinin, and several enzymes such as deoxyribonuclease and proteases have been suggested as possible virulence factors. However, several studies implicate leukotoxin, a protein cytotoxic to ruminant polymorphonuclear cells, as the major virulence factor. The importance of leukotoxin as a virulence factor in F. necrophorum infections is indicated by a correlation between toxin production and ability to induce abscesses in laboratory animals, an inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation, and a relationship between antileukotoxin antibody titers and protection against infection in experimental challenge studies.
F. necrophorum is an opportunistic pathogen that is the primary etiologic agent of liver abscesses in ruminant animals. (Scanlan, et al., (1983) Bovine rumenitis-liver abscess complex: a bacteriological review. Cornell Vet. 73:288-297; Nagaraja, T. G. et al., (1998) Liver abscesses in feedlot cattle: A review. J. Anim. Sci., 76:287-298; and Tan, et al., (1996) Fusobacterium necrophorum infections: virulence factors pathogenic mechanism and control measures. Vet. Res. Comm., 20:113-140). The organism has been recognized as an animal and human pathogen since the late 1800s, and is associated as a primary or secondary etiologic agent with numerous necrotic disease conditions in domestic and wild animals. In addition to liver abscesses, the organism is also the primary etiologic agent of foot rot, foot abscesses, calf diphtheria, and is frequently isolated from cases of mastitis, metritis, and necrotic lesions of the oral cavity.
Liver abscesses in cattle are part of a disease complex where the abscessation is secondary to primary foci of infection in the rumen epithelium. The pathogenesis can be summarized as follows: (1) ruminal lesions are induced by acidosis that follows rapid change in diet from high-roughage to high grain, prolonged feeding of high grain diet, or occasionally by foreign body penetration of the rumen epithelium; (2) bacteria present in the rumen invade the epithelium and form focal abscesses in the rumen wall; and (3) bacteria enter the portal circulation, and are carried to the liver where they localize in the parenchyma with subsequent abscess formation.
The ability of F. necrophorum to establish in the liver is attributed to the production of a toxin which is a secreted protein of high molecular weight active against leukocytes from ruminants called leukotoxin (or leucocidin). The toxin is a soluble extracellular protein that is cytotoxic to neutrophils, macrophages, hepatocytes, and ruminal cells. The leukotoxin protects against phagocytosis and is believed to aid in the establishment of F. necrophorum in the liver by directly impairing the normal defense mechanism and indirectly by the damage caused by cytolytic products released from neutrophils and macrophages to the hepatic cells. Therefore, the leukotoxin elaborated from F. necrophorum plays a critical role in F. necrophorum infection of the liver and is believed to be the primary virulence factor in the pathogenesis of liver abscesses (Tan et al., 1996).
Four biotypes (A, B, AB and C) of F. necrophorum have been described. (Langworth, (1977) Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol. Rev. 41:373-390) Biotype A, most frequently isolated from liver abscesses, is more pathogenic than biotype B, which predominates in ruminal wall abscesses. Biotypes AB and C are rarely isolated in liver abcesses (Berg, et al., (1982) Studies of Fusobacterium necrophorum from bovine hepatic abscesses: Biotypes, quantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res. 43:1580-1586), and biotype A has pathogenicity intermediate that of biotypes A and B while biotype C is non-pathogenic. (Shinjo, et al., (1990) Recognition of biovar C of Fusobacterium necrophorum (flugge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex prevot 1940) Int. J. Sys. Bacteriol. 41:395-397) Biotypes A and B, the most frequent types encountered in liver abscesses, have been assigned subspecies status: subsp. necrophorum and subsp. funduliforme, respectively (Shinjo et al., 1990). The subsp. necrophorum is more virulent, produces more leukotoxin and hemagglutinin, and is more frequently isolated from cattle liver abscesses than the subsp. funduliforme. Virulence factors and pathogenic mechanisms contributing to the formation of liver abscesses by F. necrophorum are poorly understood (Tan et al., 1996). However, several studies implicate leukotoxin to be a major virulence factor (Emery, et al., (1986) Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leukotoxin. Vet. Microbiol. 12:255-268; Tan et al., 1996). The importance of leukotoxin is evidenced by correlation between toxin production and ability to induce abscesses in laboratory animals (Coyle-Dennis, et al., (1979) Correlation between leukocidin production and virulence of two isolates of Fusobacterium necrophorum. Am. J. Vet. Res. 40:274-276; Emery and Vaughn, 1986), inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation (Emery, et al., (1985) Culture characteristics and virulence of strains of Fusobacterium necrophorum isolated from feet of cattle and sheep. Australian Vet. J. 62:43-46) and relationship between antileukotoxin antibody titers and protection in experimental challenge studies (Saginala, et al., (1996a) The serum neutralizing antibody response in cattle to Fusobacterium necrophorum leukotoxoid and possible protection against experimentally induced hepatic abscesses. Vet. Res. Comm., 20:493-504; Saginala, et al., (1996b) The serum neutralizing antibody response and protection against experimentally induced liver abscesses in steers vaccinated with Fusobacterium necrophorum. Am. J. Vet Res., 57:483-488; and Shinjo, et al., (1991) Proposal of two subspecies of Fusobacterium necrophorum (Flugge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flugge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hall 1898). Int. J. Sys. Bacteriol. 41:395-397).
Several investigators have attempted to induce protective immunity against F. necrophorum by using a variety of antigenic components. The results of such attempts have varied from ineffectual to significant protection. Clark et al. reported that cattle injected with F. necrophorum culture supernatant containing leukotoxin had a low incidence of foot rot caused by F. necrophorum. (Clark, et al. (1986), Studies into immunization of cattle against interdigital necrobacillosis. Aust. Vet. J. 63:107-110) Cell-free culture supernatant of a high leukotoxin producing strain of F. necrophorum (Tan et al., (1992) Factors affecting leukotoxin activity of F. necrophorum. Vet. Microbiol. 33:15-28), mixed with an adjuvant, was shown to elicit a high antileukotoxin antibody titer when injected in steers and provided significant protection to experimentally induced liver abscesses (Saginala et al., 1996a, b; 1997). F. necrophorum bacterin was used as an agent for immunizing cattle and sheep against liver necrosis as shown in EPO Application No. 460480 of Dec. 11, 1991 (the teachings of which are incorporated herein by reference). Specifically, virulent F. necrophorum isolates are inactivated using xcex2-propiolactone, followed by addition of adjuvants. In addition, Abe et al., Infection and Immunity, 13:1473-1478, 1976 grew F. necrophorum for 48 hours. Cells were obtained by centrifuging, washing three times with saline, and were inactivated with formalin (0.4% in saline). The inactivated cells were then injected into mice to induce immunity. Two weeks after the last booster injection, each mouse was challenged with viable cells of F. necrophorum. The mice immunized with killed cells and challenged with live cells had no detectable bacteria in the liver, lung or spleen for up to 28 days. It was concluded that immunization of mice with formalin-killed F. necrophorum conferred protection against infection. Garcia et al., (Canadian J. Comp. Med, 38:222-226, 1974), conducted field trials to evaluate the efficacy of alum-precipitated toxoids of F. necrophorum. The vaccine preparation consisted of washed cells (unlikely to contain leukotoxin) that were ruptured by sonication. The most promising result was achieved with the injection of 15.5 mg protein of cytoplasmic toxoid. In this group, the incidents of liver abscesses was reduced to 10% from an average 35% in the control group. Emery et al., Vet. Microbiol., 12:255-268, 1986, prepared material by gel filtration of 18-hour culture supernate of F. necrophorum. This elicited significant immunity against challenge by with viable F. necrophorum. The injected preparation contained endotoxin and the majority of the leukotoxic activity. U.S. Pat. No. 5,455,034 (the teachings of which are incorporated herein by reference) by Nagaraja et al. disclosed that prevention of leukotoxin production (or inhibition of its activity) in immunized animals prevents the establishment of F. necrophorum infection. Thus, immunization of the animals against F. necrophorum leukotoxin, so that the animals"" white blood cells or tissue macrophages may phagocytize the bacteria, presented a way to prevent diseases associated with F. necrophorum infection, e.g., liver abscesses in cattle and sheep, and foot rot in cattle. In order to produce such a leukotoxoid vaccine, the F. necrophorum bacteria was cultured in away to enhance the elaboration of leukotoxin in the supernate. Thereupon, bacterial growth and leukotoxin elaboration was terminated, and a vaccine prepared by inactivating at least the leukotoxin-containing supernate. In more detail, the leukotoxin elaboration method of the ""034 patent involved first forming a culture of F. necrophorum bacteria in growth media, and thereafter causing the bacteria to grow in the culture and to simultaneously elaborate leukotoxin in the supernate. At the end of the culturing step, i.e., at the end of the selected culture time within the range of from about 4-10 hours, the bacterial growth and leukotoxin elaboration were terminated, and the leukotoxoid vaccine was prepared. This involved first separating the leukotoxin-containing supernate from the bacteria, followed by inactivation through use of formalin, xcex2-propiolactone, heat, radiation or any other known method of inactivation. Alternately, the entire culture could be inactivated to form the vaccine.
Presently, the control of liver abscesses is with the use of antimicrobial feed additives. Antimicrobial compounds reduce the incidence of liver abscesses but do not eliminate the problem (Nagaraja et al., 1998). Therefore, an effective vaccine would be highly desirable to the feedlot industry. The vaccine approach also would alleviate public health concerns associated with the use of subtherapeutic levels of antibiotics in the feed. Because studies have indicated that antileukotoxin immunity reduces the incidence of hepatic abscesses and interdigital necrobacillosis (Garcia et al., 1974; Clark et al., 1986; Saginala et al., 1996a, b; 1997), the development of a recombinant leukotoxin vaccine will be of great value in the control of hepatic and interdigital necrobacillosis in cattle.
In order to better define the molecular nature of the F. necrophorum leukotoxin, and as a first step toward determining its specific role in the virulence of this bacterium, the leukotoxin gene was isolated, its nucleotide sequence determined, and the recombinant leukotoxin was expressed in E. coli. 
The leukotoxin open reading frame (lktA) is part of a multi-gene operon containing 9,726 bp, and encoding a protein containing 3,241 amino acids with an overall molecular weight of 335,956 daltons. F. necrophorum leukotoxin is highly unstable as evidenced by western blot analysis of native leukotoxin (culture supernatant, sephadex gel or affinity purified) (FIG. 1). In this Figure, lane 1 contains whole cell lysate of E. coli cells expressing full-length recombinant leukotoxin, lane 2 contains Immuno-affinity purified native leukotoxin, lane 3 contains Sephadex gel purified leukotoxin, and lane 4 contains culture supernatant from F. necrophorum concentrated 60 times. The blots were probed with polyclonal antiserum raised in rabbits against affinity purified native leukotoxin. Because of the apparent instability of the full-length recombinant leukotoxin protein, the protein encoded by the gene was truncated into five recombinant polypeptides (or protein fragments, BSBSE, SX, GAS, SH and FINAL) having overlapping regions by truncating the full length gene into five different sections and amplifying, expressing in E. coli, and recovering the protein or polypeptide encoded by each of these sections. These polypeptides along with the full length protein are then tested to determine their immunogenicity and protective immunity in comparison to the efficacy of immunization conferred by inactivated native leukotoxin in F. necrophorum culture supernatant.
Specifically, the chromosomal DNA was extracted from F. necrophorum and partially digested by restriction endonucleases prior to being size-fractionated by sucrose gradient centrifugation. The 10-12 kb fragments were then ligated into a BamHI digested, dephosphorylated xcexZAP expression vector. Recombinant phages were infected into Escherichia coli and plated onto agar plates. Plaque lifts were performed (with polyclonal antiserum raised in rabbits against affinity purified leukotoxin) using an immunoscreening kit. Six immunoreactive recombinant phages were identified and denominated as clones 816, 611, 513, 911, 101, and 103. These clones were plaque-purified three times to ensure purity, phagemids rescued, and anti-leukotoxin immunoreactivity of the encoded proteins was confirmed. This immunoreactivity verified that the clones represented native leukotoxin F. necrophorum. 
Expression of a polypeptide encoded by the 3.5 kb from the 5xe2x80x2 end of the lktA caused immediate cessation of the growth and lysis of E. coli host cells suggesting that regions of leukotoxin could be toxic to E. coli. Of course, the objective was to create overlapping gene truncations extending over the entire lktA ORF so that the resulting polypeptide products are small and relatively stable on expression, but are large enough to be immunogenic. Also, the effectiveness of various recombinant truncated leukotoxin polypeptides alone or in combinations as immunogens and evaluated protective immunity against challenge with F. necrophorum in mice was investigated. The use of mice as an experimental model for F. necrophorum infection in cattle is well established (Abe et al., 1976; Conion et al., 1977; Smith et al., 1989; Garcia and McKay, 1978; Emery and Vaughan, 1986). Extension of the patterns of immunity and infection to cattle has shown that mice can be a valuable model to evaluate the immunogenicity and protection provided by various F. necrophorum fractions (Garcia et al., 1975; Garcia and McKay, 1978). Studies have also indicated that strains of F. necrophorum that are pathogenic in domestic animals, frequently are pathogenic in mice suggesting necrobacillosis as a disease is similar among these species of animals (Smith and Thornton, 1993).
The nucleotide sequence of the full length version of the gene is designated as SEQ ID No. 8 and the nucleotide sequences of the five truncations of the full length gene are designated as BSBSE (SEQ ID No. 9), SX (SEQ ID No. 10), GAS (SEQ ID No. 11), SH (SEQ ID No. 12), and FINAL (SEQ ID No. 13). Additionally, the nucleotide sequence of the upstream region of the full length gene is designated UPS (SEQ ID No. 14). The amino acid sequence of the full length protein encoded by the F. necrophorum gene is designated as SEQ ID No. 1 and the amino acid sequences of the truncated protein fragments respectively encoded by BSBSE, SX, GAS, SH and FINAL are designated as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6. In the case of UPS, the polypeptide or truncated protein fragment encoded for by UPS is designated as SEQ ID No. 7. Finally, SEQ ID No. 15 is the fall length gene sequence along with contiguous sequences.
Truncated recombinant polypeptides were purified by nickel affinity chromatography, and injected into rabbits to raise polyclonal antisera. Antibodies raised against two of the five polypeptides (BSBSE and GAS) neutralized the toxicity of F. necrophorum leukotoxin against bovine neutrophils. The effectiveness of the purified truncated polypeptides to induce a protective immunity was determined by injecting the polypeptides, individually or in mixtures, homogenized with Ribi adjuvant in mice, followed by experimental challenge with F. necrophorum. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection and the extent of protection was greater than the full-length native leukotoxin or inactivated culture supernatant. The study provided further credence to the importance of leukotoxin as the major virulence factor of F. necrophorum and the protein carries a domain(s) or epitope(s) that induces protective immunity against experimental infection.
The DNA and deduced amino acid sequences were compared with sequences in Genbank but no significant similarities (no sequences having greater than 22% sequence identity) were found. Thus, the F. necrophorum leukotoxin appears to be distinct from all known leukotoxins and RTX-type toxins. When the deduced amino acid sequence of the lktA region was subjected to the Kyte-Doolittle hydropathy analysis (FIG. 3), 14 sites of sufficient length and hydrophobic character to be potential membrane spanning regions, were found. Upstream to the leukotoxin ORF is an open reading frame of at least 1.4 kb in length, which is in the same orientation. It encodes a protein that has significant sequence similarity (21% or 62 out of 283 residues) to the heme-hemopexin utilization protein (UxuB) of Haemophilus infuenzae. 
Bacterial leukotoxins and cytotoxins generally have molecular masses of less than 200 kDa. This includes characterized leukotoxins of Pasteurella hemolytica (104,000 kDa; 10), Staphylococcus aureus (38,000+32,000 kDa; 20), or Actinomyces actinomycetecomitans (114,000 kDa; 15) or other pore-forming toxins of gram-negative bacteria (103,000to 198,000 kDa; 30). However, leukotoxin secreted by F. necrophorum was shown to be approximately 300 kDa in size based on sephadex column purification and SDS-PAGE analyses.
As used herein, the following definitions will apply: xe2x80x9cSequence Identityxe2x80x9d as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are xe2x80x9cidenticalxe2x80x9d at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et al., eds., M. Stockton Press, New York (1991); and Carillo, H., et al. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol.,215:403-410(1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410(1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% xe2x80x9csequence identityxe2x80x9d to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence maybe deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence maybe inserted into the reference sequence. These mutations of the reference sequence may occur at the 5xe2x80x2 or 3xe2x80x2 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example,95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence maybe deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, xe2x80x9csequence homologyxe2x80x9d, as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary. However, in contrast to xe2x80x9csequence identityxe2x80x9d, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence maybe inserted into the reference sequence.
A xe2x80x9cconservative substitutionxe2x80x9d refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
Isolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not xe2x80x9cisolated,xe2x80x9d but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein. Finally, all references and teachings cited herein which have not been expressly incorporated by reference are hereby incorporated by reference.
Preferably, sequences having at least about 50% sequence homology or at least about 60% sequence identity with any of SEQ ID Nos. 1-15 are used for purposes of the present invention. More preferably, sequences having at least about 60% sequence homology or at least about 70% sequence identity are used for purposes of the present invention. Still more preferably, sequences having at least about 75% sequence homology or at least about 85% sequence identity are used for purposes of the present invention. Even more preferably, sequences having at least about 87% sequence homology or at least about 92% sequence identity are used for purposes of the present invention. Most preferably, sequences having at least about 95% sequence homology or at least about 98% sequence identity are used for purposes of the present invention.